The structural glycoprotein E (an envelope protein with RNase activity) of classical swine fever virus (CSFV) is not well characterized with respect to its antigenic structure and organization. Here, we investigated the antigenic sites on E by raising mAbs against the expressed E of CSFV strain Alfort/187 and defined the B-cell epitopes recognized by these antibodies. Eighteen mAbs to E were identified and they were classified as either immunoglobulin subclass G1 or G2b. Using an array of overlapping 12-mer peptides, spanning aa 27–227 of E, the epitopes for 12 mAbs were mapped to a high resolution of six to eight residues, which cluster in five discrete locations, GIWPEKIC (group I), NYTCCKLQ (group II), QARNRPTT (group III), SFAGTVIE (group IV) and VEDILY (group V). Two mAbs recognize two or more antigenic determinants, including the group II epitope. The epitopes for four other mAbs could not be mapped using the overlapping 12-mer peptides. Random peptide phage display with one mAb from each of all the groups except group V further identified some conserved residues that may be critical for binding antibodies, i.e. Trp in the epitope of group I, Leu in the epitope of group II, Gln and Apn in the epitope of group III, and Ser and Gly in the epitope of group IV. This study has provided new insights into the structure and organization of epitopes on the CSFV E and valuable epitope information for the rational design of vaccines, drugs and diagnostic immunoassays for CSFV.


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  1. Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, F. R., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A. & other authors(1984). The antigenic structure of proteins: a reappraisal. Annu Rev Immunol 2, 67–101.[CrossRef] [Google Scholar]
  2. Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef] [Google Scholar]
  3. Bruschke, C. J., Hulst, M. M., Moormann, R. J., van Rijn, P. A. & van Oirschot, J. T.(1997). Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. J Virol 71, 6692–6696. [Google Scholar]
  4. Christmann, A., Wentzel, A., Meyer, C., Meyers, G. & Kolmar, H.(2001). Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries. J Immunol Methods 257, 163–173.[CrossRef] [Google Scholar]
  5. Clavijo, A., Lin, M., Riva, J., Mallory, M., Lin, F. & Zhou, E. M.(2001). Development of a competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus. Res Vet Sci 70, 1–7.[CrossRef] [Google Scholar]
  6. de Smit, A. J., Bouma, A., de Kluijver, E. P., Terpstra, C. & Moormann, R. J.(2001). Duration of the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination. Vet Microbiol 78, 307–317.[CrossRef] [Google Scholar]
  7. Hilpert, K., Winkler, D. F. & Hancock, R. E.(2007). Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat Protoc 2, 1333–1349.[CrossRef] [Google Scholar]
  8. Horiuchi, H., Yanai, K., Takagi, M., Yano, K., Wakabayashi, E., Sanda, A., Mine, S., Ohgi, K. & Irie, M.(1988). Primary structure of a base non-specific ribonuclease from Rhizopus niveus. J Biochem 103, 408–418. [Google Scholar]
  9. Hulst, M. M. & Moormann, R. J.(1997). Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors. J Gen Virol 78, 2779–2787. [Google Scholar]
  10. Hulst, M. M., Westra, D. F., Wensvoort, G. & Moormann, R. J.(1993). Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 67, 5435–5442. [Google Scholar]
  11. Hulst, M. M., Himes, G., Newbigin, E. & Moormann, R. J.(1994). Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology 200, 558–565.[CrossRef] [Google Scholar]
  12. Hulst, M. M., Panoto, F. E., Hoekman, A., van Gennip, H. G. & Moormann, R. J.(1998). Inactivation of the RNase activity of glycoprotein Erns of classical swine fever virus results in a cytopathogenic virus. J Virol 72, 151–157. [Google Scholar]
  13. Hulst, M. M., van Gennip, H. G. & Moormann, R. J.(2000). Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J Virol 74, 9553–9561.[CrossRef] [Google Scholar]
  14. Hulst, M. M., van Gennip, H. G., Vlot, A. C., Schooten, E., de Smit, A. J. & Moormann, R. J.(2001). Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence. J Virol 75, 9585–9595.[CrossRef] [Google Scholar]
  15. König, M., Lengsfeld, T., Pauly, T., Stark, R. & Thiel, H.-J.(1995). Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. J Virol 69, 6479–6486. [Google Scholar]
  16. Kosmidou, A., Ahl, R., Thiel, H. J. & Weiland, E.(1995). Differentiation of classical swine fever virus (CSFV) strains using monoclonal antibodies against structural glycoproteins. Vet Microbiol 47, 111–118.[CrossRef] [Google Scholar]
  17. Kurihara, H., Nonaka, T., Mitsui, Y., Ohgi, K., Irie, M. & Nakamura, K. T.(1996). The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 Å resolution. J Mol Biol 255, 310–320.[CrossRef] [Google Scholar]
  18. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef] [Google Scholar]
  19. Langedijk, J. P., Middel, W. G., Meloen, R. H., Kramps, J. A. & de Smit, J. A.(2001). Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein Erns for serologic diagnosis of pestivirus infections in swine. J Clin Microbiol 39, 906–912.[CrossRef] [Google Scholar]
  20. Langedijk, J. P., van Veelen, P. A., Schaaper, W. M., de Ru, A. H., Meloen, R. H. & Hulst, M. M.(2002). A structural model of pestivirus Erns based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide. J Virol 76, 10383–10392.[CrossRef] [Google Scholar]
  21. Lin, M., Lin, F., Mallory, M. & Clavijo, A.(2000). Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol 74, 11619–11625.[CrossRef] [Google Scholar]
  22. Lin, M., Trottier, E., Pasick, J. & Sabara, M.(2004). Identification of antigenic regions of the Erns protein for pig antibodies elicited during classical swine fever virus infection. J Biochem 136, 795–804.[CrossRef] [Google Scholar]
  23. Lin, M., Trottier, E. & Mallory, M.(2005a). Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection. Clin Diagn Lab Immunol 12, 877–881. [Google Scholar]
  24. Lin, M., Trottier, E. & Pasick, J.(2005b). Antibody responses of pigs to defined Erns fragments after infection with classical swine fever virus. Clin Diagn Lab Immunol 12, 180–186. [Google Scholar]
  25. Lin, M., Todoric, D., Mallory, M., Luo, B. S., Trottier, E. & Dan, H.(2006). Monoclonal antibodies binding to the cell surface of Listeria monocytogenes serotype 4b. J Med Microbiol 55, 291–299.[CrossRef] [Google Scholar]
  26. Liu, S., Tu, C., Wang, C., Yu, X., Wu, J., Guo, S., Shao, M., Gong, Q., Zhu, Q. & Kong, X.(2006a). The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2. J Virol Methods 134, 125–129.[CrossRef] [Google Scholar]
  27. Liu, S., Yu, X., Wang, C., Wu, J., Kong, X. & Tu, C.(2006b). Quadruple antigenic epitope peptide producing immune protection against classical swine fever virus. Vaccine 24, 7175–7180.[CrossRef] [Google Scholar]
  28. Matsuura, T., Sakai, H., Unno, M., Ida, K., Sato, M., Sakiyama, F. & Norioka, S.(2001). Crystal structure at 1.5 Å resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility. J Biol Chem 276, 45261–45269.[CrossRef] [Google Scholar]
  29. Meyers, G., Saalmuller, A. & Buttner, M.(1999). Mutations abrogating the RNase activity in glycoprotein Erns of the pestivirus classical swine fever virus lead to virus attenuation. J Virol 73, 10224–10235. [Google Scholar]
  30. Moennig, V.(1988). Characteristics of the virus. In Classical Swine Fever and Related Viral Infections, pp. 55–58. Edited by Liess, B.. Boston, MA. : Martinus Nijhoff Publishing. [Google Scholar]
  31. Moser, C., Ruggli, N., Tratschin, J. D. & Hofmann, M. A.(1996). Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2. Vet Microbiol 51, 41–53.[CrossRef] [Google Scholar]
  32. Müller, A., Depner, K. R. & Liess, B.(1996). Evaluation of a gp 55 (E2) recombinant-based ELISA for the detection of antibodies induced by classical swine fever virus. Dtsch Tierarztl Wochenschr 103, 451–453. [Google Scholar]
  33. Nakagawa, A., Tanaka, I., Sakai, R., Nakashima, T., Funatsu, G. & Kimura, M.(1999). Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 Å resolution. Biochim Biophys Acta 1433, 253–260.[CrossRef] [Google Scholar]
  34. Oganesyan, N., Kim, S. H. & Kim, R.(2005). On-column protein refolding for crystallization. J Struct Funct Genomics 6, 177–182.[CrossRef] [Google Scholar]
  35. Rice, M. C. & Lindenbach, B. D.(2001).Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 991–1041. Edited by Knipe, D. M. & Howley, P. M.. Philadelphia. : Lippincott Williams & Wilkins. [Google Scholar]
  36. Risatti, G. R., Holinka, L. G., Carrillo, C., Kutish, G. F., Lu, Z., Tulman, E. R., Sainz, I. F. & Borca, M. V.(2006). Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus. Virology 355, 94–101.[CrossRef] [Google Scholar]
  37. Ruggli, N., Moser, C., Mitchell, D., Hofmann, M. & Tratschin, J. D.(1995). Baculovirus expression and affinity purification of protein E2 of classical swine fever virus strain Alfort/187. Virus Genes 10, 115–126.[CrossRef] [Google Scholar]
  38. Rümenapf, T., Unger, G., Strauss, J. H. & Thiel, H.-J.(1993). Processing of the envelope glycoproteins of pestiviruses. J Virol 67, 3288–3294. [Google Scholar]
  39. Sainz, I. F., Holinka, L. G., Lu, Z., Risatti, G. R. & Borca, M. V.(2008). Removal of a N-linked glycosylation site of classical swine fever virus strain Brescia Erns glycoprotein affects virulence in swine. Virology 370, 122–129.[CrossRef] [Google Scholar]
  40. Schein, C. H.(1997). From housekeeper to microsurgeon: the diagnostic and therapeutic potential of ribonucleases. Nat Biotechnol 15, 529–536.[CrossRef] [Google Scholar]
  41. Schneider, R., Unger, G., Stark, R., Schneider-Scherzer, E. & Thiel, H. J.(1993). Identification of a structural glycoprotein of an RNA virus as a ribonuclease. Science 261, 1169–1171.[CrossRef] [Google Scholar]
  42. Shulman, M., Wilde, C. D. & Kohler, G.(1978). A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269–270.[CrossRef] [Google Scholar]
  43. Thiel, H. J., Stark, R., Weiland, E., Rümenapf, T. & Meyers, G.(1991). Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65, 4705–4712. [Google Scholar]
  44. van Rijn, P. A., van Gennip, R. G., de Meijer, E. J. & Moormann, R. J.(1992). A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Vet Microbiol 33, 221–230.[CrossRef] [Google Scholar]
  45. van Rijn, P. A., van Gennip, H. G., de Meijer, E. J. & Moormann, R. J.(1993). Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia. J Gen Virol 74, 2053–2060.[CrossRef] [Google Scholar]
  46. van Rijn, P. A., Miedema, G. K., Wensvoort, G., van Gennip, H. G. & Moormann, R. J.(1994). Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J Virol 68, 3934–3942. [Google Scholar]
  47. van Rijn, P. A., Bossers, A., Wensvoort, G. & Moormann, R. J.(1996). Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J Gen Virol 77, 2737–2745.[CrossRef] [Google Scholar]
  48. Weiland, E., Stark, R., Haas, B., Rümenapf, T., Meyers, G. & Thiel, H. J.(1990). Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol 64, 3563–3569. [Google Scholar]
  49. Weiland, E., Ahl, R., Stark, R., Weiland, F. & Thiel, H. J.(1992). A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus. J Virol 66, 3677–3682. [Google Scholar]
  50. Wengler, G.(1991). Classification and nomenclature of viruses. Fifth report of the international committee on taxonomy of viruses. Arch Virol Suppl 2, 223–233. [Google Scholar]
  51. Wensvoort, G.(1989). Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies. J Gen Virol 70, 2865–2876.[CrossRef] [Google Scholar]
  52. Windisch, J. M., Schneider, R., Stark, R., Weiland, E., Meyers, G. & Thiel, H. J.(1996). RNase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J Virol 70, 352–358. [Google Scholar]
  53. Winkler, D. F. & Campbell, W. D.(2008). The spot technique: synthesis and screening of peptide macroarrays on cellulose membranes. Methods Mol Biol 494, 47–70. [Google Scholar]
  54. Yu, M., Wang, L. F., Shiell, B. J., Morrissy, C. J. & Westbury, H. A.(1996). Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses. Virology 222, 289–292.[CrossRef] [Google Scholar]
  55. Zhang, F., Yu, M., Weiland, E., Morrissy, C., Zhang, N., Westbury, H. & Wang, L. F.(2006). Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library. Arch Virol 151, 37–54.[CrossRef] [Google Scholar]
  56. Zola, H.(1987).Monoclonal Antibodies: a Manual of Techniques. Boca Raton, FL. : CRC Press. [Google Scholar]

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